Ubiquitin is a small protein of only 76 amino acids that functions as a post-translational modification for other proteins. Ubiquitin is one of the most highly conserved proteins known. The past decade has seen an upsurge in knowledge about the ubiquitin pathway, as well as the identification of several ubiquitin-like pathways, and a Nobel Prize awarded to ubiquitin pioneers. Yet, there is still much that remains to be learned about this important pathway. Within the genomes of eukaryotic organisms a large number of ubiquitin pathway enzymes and ubiquitin binding proteins exist. The proposed research is aimed at understanding the diverse roles of ubiquitin pathway enzymes with an emphasis on elucidating tissue specific functions. The nematode, C. elegans, is well-suited as a model for these studies because of copious genetic resources and excellent cell biology techniques. Ubiquitin from C. elegans differs by only one amino acid from humans. Previous studies into the ubiquitin pathway in C. elegans identified more than 20 ubiquitin conjugating enzymes (Ubc) and greater than a hundred ubiquitin ligases (E3). Initial RNAi studies of these enzymes illuminated the function of a few Ubcs, however many well-conserved Ubcs exhibited no RNAi phenotype. Preliminary studies now indicate substantial overlap in Ubc function. RNAi with pools of four or five Ubcs can result in a phenotype not seen when RNAi is done with each of the Ubcs individually. Specifically, six out of 85 pools tested exhibited a strongly penetrant sterile phenotype. Specific Aim #1 in this proposal will extend these preliminary studies with a careful phenotypic analysis of the mutant phenotypes resulting from RNAi with the Ubc pools. Specific Aim #2 proposes to study the tissue specific expression of different Ubcs by means of reporter genes linked to Ubc promoters. In Specific Aim #3, protein profiling of ubiquitinated proteins in different tissues will be accomplished via tissue specific expression of a tagged ubiquitin. Ubiquitination profiles will be matched to data on the tissue specific expression of individual Ubcs. This profiling will provide a better understanding of how the ubiquitin pathway differs between different tissues. The achievement of these aims will provide important information about the ubiquitin pathway and will cultivate tools for future ubiquitin studies. Since ubiquitin enzymes have been implicated in several diseases, it is important to understand the functions of the various enzymes and the interactions that these enzymes have within the organism.